Fragments of HdhQ150 Mutant Huntingtin Form a Soluble Oligomer Pool That Declines with Aggregate Deposition upon Aging

Cleavage of the full-length mutant huntingtin (mhtt) protein into smaller, soluble aggregation-prone mhtt fragments appears to be a key process in the neuropathophysiology of Huntington’s Disease (HD). Recent quantification studies using TR-FRET-based immunoassays showed decreasing levels of soluble mhtt correlating with an increased load of aggregated mhtt in the aging HdhQ150 mouse brain. To better characterize the nature of these changes at the level of native mhtt species, we developed a detection method that combines size exclusion chromatography (SEC) and time-resolved fluorescence resonance energy transfer (TR-FRET) that allowed us to resolve and define the formation, aggregation and temporal dynamics of native soluble mhtt species and insoluble aggregates in the brain of the HdhQ150 knock-in mouse. We found that mhtt fragments and not full-length mhtt form oligomers in the brains of one month-old mice long before disease phenotypes and mhtt aggregate histopathology occur. As the HdhQ150 mice age, brain levels of soluble full-length mhtt protein remain similar. In contrast, the soluble oligomeric pool of mhtt fragments slightly increases during the first two months before it declines between 3 and 8 months of age. This decline inversely correlates with the formation of insoluble mhtt aggregates. We also found that the pool-size of soluble mhtt oligomers is similar in age-matched heterozygous and homozygous HdhQ150 mouse brains whereas insoluble aggregate formation is greatly accelerated in the homozygous mutant brain. The capacity of the soluble mhtt oligomer pool therefore seems exhausted already in the heterozygous state and likely kept constant by changes in flux and, as a consequence, increased rate of insoluble aggregate formation. We demonstrate that our novel findings in mice translate to human HD brain but not HD patient fibroblasts.     

Aggregation Properties of the Small Nuclear Ribonucleoprotein U1-70K in Alzheimer Disease

Recent evidence indicates that U1-70K and other U1 small nuclear ribonucleoproteins are Sarkosyl-insoluble and associate with Tau neurofibrillary tangles selectively in Alzheimer disease (AD). Currently, the mechanisms underlying the conversion of soluble nuclear U1 small nuclear ribonucleoproteins into insoluble cytoplasmic aggregates remain elusive. Based on the biochemical and subcellular distribution properties of U1-70K in AD, we hypothesized that aggregated U1-70K itself or other biopolymers (e.g. proteins or nucleic acids) interact with and sequester natively folded soluble U1-70K into insoluble aggregates. Here, we demonstrate that total homogenates from AD brain induce soluble U1-70K from control brain or recombinant U1-70K to become Sarkosyl-insoluble. This effect was not dependent on RNA and did not correlate with detergent-insoluble Tau levels as AD homogenates with reduced levels of these components were still capable of inducing U1-70K aggregation. In contrast, proteinase K-treated AD homogenates and Sarkosyl-soluble AD fractions were unable to induce U1-70K aggregation, indicating that aggregated proteins in AD brain are responsible for inducing soluble U1-70K aggregation. It was determined that the C terminus of U1-70K, which harbors two disordered low complexity (LC) domains, is necessary for U1-70K aggregation. Moreover, both LC1 and LC2 domains were sufficient for aggregation. Finally, protein cross-linking and mass spectrometry studies demonstrated that a U1-70K fragment harboring the LC1 domain directly interacts with aggregated U1-70K in AD brain. Our results support a hypothesis that aberrant forms of U1-70K in AD can directly sequester soluble forms of U1-70K into insoluble aggregates.     

Regulation of BCL-X splicing reveals a role for the polypyrimidine tract binding protein (PTBP1/hnRNP I) in alternative 5′ splice site selection

Alternative splicing (AS) modulates many physiological and pathological processes. For instance, AS of the BCL-X gene balances cell survival and apoptosis in development and cancer. Herein, we identified the polypyrimidine tract binding protein (PTBP1) as a direct regulator of BCL-X AS. Overexpression of PTBP1 promotes selection of the distal 5′ splice site in BCL-X exon 2, generating the pro-apoptotic BCL-Xs splice variant. Conversely, depletion of PTBP1 enhanced splicing of the anti-apoptotic BCL-XL variant. In vivo cross-linking experiments and site-directed mutagenesis restricted the PTBP1 binding site to a polypyrimidine tract located between the two alternative 5′ splice sites. Binding of PTBP1 to this site was required for its effect on splicing. Notably, a similar function of PTBP1 in the selection of alternative 5′ splice sites was confirmed using the USP5 gene as additional model. Mechanistically, PTBP1 displaces SRSF1 binding from the proximal 5′ splice site, thus repressing its selection. Our study provides a novel mechanism of alternative 5′ splice site selection by PTBP1 and indicates that the presence of a PTBP1 binding site between two alternative 5′ splice sites promotes selection of the distal one, while repressing the proximal site by competing for binding of a positive regulator.     

Widespread Protein Aggregation as an Inherent Part of Aging in C. elegans

Aberrant protein aggregation is a hallmark of many age-related diseases, yet little is known about whether proteins aggregate with age in a non-disease setting. Using a systematic proteomics approach, we identified several hundred proteins that become more insoluble with age in the multicellular organism Caenorhabditis elegans. These proteins are predicted to be significantly enriched in β-sheets, which promote disease protein aggregation. Strikingly, these insoluble proteins are highly over-represented in aggregates found in human neurodegeneration. We examined several of these proteins in vivo and confirmed their propensity to aggregate with age. Different proteins aggregated in different tissues and cellular compartments. Protein insolubility and aggregation were significantly delayed or even halted by reduced insulin/IGF-1-signaling, which also slows aging. We found a significant overlap between proteins that become insoluble and proteins that influence lifespan and/or polyglutamine-repeat aggregation. Moreover, overexpressing one aggregating protein enhanced polyglutamine-repeat pathology. Together our findings indicate that widespread protein insolubility and aggregation is an inherent part of aging and that it may influence both lifespan and neurodegenerative disease.     

Alternative splicing regulation at tandem 3' splice sites

Alternative splicing (AS) constitutes a major mechanism creating protein diversity in humans. Previous bioinformatics studies based on expressed sequence tag and mRNA data have identified many AS events that are conserved between humans and mice. Of these events, ~25% are related to alternative choices of 3′ and 5′ splice sites. Surprisingly, half of all these events involve 3′ splice sites that are exactly 3 nt apart. These tandem 3′ splice sites result from the presence of the NAGNAG motif at the acceptor splice site, recently reported to be widely spread in the human genome. Although the NAGNAG motif is common in human genes, only a small subset of sites with this motif is confirmed to be involved in AS. We examined the NAGNAG motifs and observed specific features such as high sequence conservation of the motif, high conservation of ~30 bp at the intronic regions flanking the 3′ splice site and overabundance of cis-regulatory elements, which are characteristic of alternatively spliced tandem acceptor sites and can distinguish them from the constitutive sites in which the proximal NAG splice site is selected. Our findings imply that AS at tandem splice sites and constitutive splicing of the distal NAG are highly regulated.     

Orthogonal cross-seeding: an approach to explore protein aggregates in living cells

Protein aggregation is associated with the pathology of many diseases, especially neurodegenerative diseases. A variety of structurally polymorphic aggregates or preaggregates including amyloid fibrils is accessible to any aggregating protein. Preaggregates are now believed to be the toxic culprits in pathologies rather than mature aggregates. Although clearly valuable, understanding the mechanism of formation and the structural characteristics of these prefibrillar species is currently lacking. We report here a simple new approach to map the nature of the aggregate core of transient aggregated species directly in the cell. The method is conceptually based on the highly discriminating ability of aggregates to recruit new monomeric species with equivalent molecular structure. Different soluble segments comprising parts of an amyloidogenic protein were transiently pulse-expressed in a tightly controlled, time-dependent manner along with the parent aggregating full-length protein, and their recruitment into the insoluble aggregate was monitored immunochemically. We used this approach to determine the nature of the aggregate core of the metastable aggregate species formed during the course of aggregation of a chimera containing a long polyglutamine repeat tract in a bacterial host. Strikingly, we found that different segments of the full-length protein dominated the aggregate core at different times during the course of aggregation. In its simplicity, the approach is also potentially amenable to screen also for compounds that can reshape the aggregate core and induce the formation of alternative nonamyloidogenic species.     

Generation,Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer''s disease (Aβ, tau), Parkinson''s Disease (α-synuclein), Huntington''s disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease^{1, 2}, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia³. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C⁴, and that DISC1 aggresomes generated in vitro were cell-invasive⁵, similar to what had been shown for Aβ⁶, tau^7-9, α-synuclein¹⁰, polyglutamine¹¹, or SOD1 aggregates¹². These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders. Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic α-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.     

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (α-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, α-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated α-syn is a neuropathological feature of Parkinson''s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble α-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of α-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated α-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal α-syn biology in transgenic animal models harbouring different α-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.     

Proteinaceous infectious behavior in non-pathogenic proteins is controlled by molecular chaperones

External stresses or mutations may cause labile proteins to lose their distinct native conformations and seek alternatively stable aggregated forms. Molecular chaperones that specifically act on protein aggregates were used here as a tool to address the biochemical nature of stable homo- and hetero-aggregates from non-pathogenic proteins formed by heat-stress. Confirmed by sedimentation and activity measurements, chaperones demonstrated that a single polypeptide chain can form different species of aggregates, depending on the denaturing conditions. Indicative of a cascade reaction, sub-stoichiometric amounts of one fast-aggregating protein strongly accelerated the conversion of another soluble, slow-aggregating protein into insoluble, chaperone-resistant aggregates. Chaperones strongly inhibited seed-induced protein aggregation, suggesting that they can prevent and cure proteinaceous infectious behavior in homo- and hetero-aggregates from common and disease-associated proteins in the cell.     

Proteins that mediate protein aggregation and cytotoxicity distinguish Alzheimer's hippocampus from normal controls

Neurodegenerative diseases are distinguished by characteristic protein aggregates initiated by disease‐specific ‘seed’ proteins; however, roles of other co‐aggregated proteins remain largely unexplored. Compact hippocampal aggregates were purified from Alzheimer's and control‐subject pools using magnetic‐bead immunoaffinity pulldowns. Their components were fractionated by electrophoretic mobility and analyzed by high‐resolution proteomics. Although total detergent‐insoluble aggregates from Alzheimer's and controls had similar protein content, within the fractions isolated by tau or Aβ_1–42 pulldown, the protein constituents of Alzheimer‐derived aggregates were more abundant, diverse, and post‐translationally modified than those from controls. Tau‐ and Aβ‐containing aggregates were distinguished by multiple components, and yet shared >90% of their protein constituents, implying similar accretion mechanisms. Alzheimer‐specific protein enrichment in tau‐containing aggregates was corroborated for individuals by three analyses. Five proteins inferred to co‐aggregate with tau were confirmed by precise in situ methods, including proximity ligation amplification that requires co‐localization within 40 nm. Nematode orthologs of 21 proteins, which showed Alzheimer‐specific enrichment in tau‐containing aggregates, were assessed for aggregation‐promoting roles in C. elegans by RNA‐interference ‘knockdown’. Fifteen knockdowns (71%) rescued paralysis of worms expressing muscle Aβ, and 12 (57%) rescued chemotaxis disrupted by neuronal Aβ expression. Proteins identified in compact human aggregates, bound by antibody to total tau, were thus shown to play causal roles in aggregation based on nematode models triggered by Aβ_1–42. These observations imply shared mechanisms driving both types of aggregation, and/or aggregate‐mediated cross‐talk between tau and Aβ. Knowledge of protein components that promote protein accrual in diverse aggregate types implicates common mechanisms and identifies novel targets for drug intervention.     

Elevated synthesis of human alpha 1-antitrypsin hinders the secretion of murine alpha 1-antitrypsin from hepatocytes of transgenic mice

alpha 1-Antitrypsin (AAT) is a major hepatic secretory protein. The elevated synthesis of human AAT within hepatocytes of transgenic mice results in its accumulation within a subset of distended cisternae of the rough endoplasmic reticulum. The protein does not accumulate in large insoluble aggregates as is the case for the human PiZ AAT variant. Furthermore, the accumulated protein is not associated with immunoglobulin heavy chain binding protein. Transgenic animals exhibiting an elevated synthesis and subsequent intrahepatic accumulation of human AAT exhibit reduced serum levels of murine AAT as a result of its hindered secretion and accumulation within the rough endoplasmic reticulum. Interestingly, the secretion of murine transferrin and albumin which represent glycosylated and non-glycosylated hepatic secretory proteins, respectively, is unaffected. Overall, these results demonstrate that the elevated synthesis of human AAT can hinder the export of murine AAT from the hepatic rough endoplasmic reticulum in an apparently specific manner.     

Rapid assembly of amyloid-beta peptide at a liquid/liquid interface produces unstable beta-sheet fibers

Accumulation of aggregated amyloid-beta peptide (Abeta) in the brain is a pathological hallmark of Alzheimer's disease (AD). In vitro studies indicate that the 40- to 42-residue Abeta peptide in solution will undergo self-assembly leading to the transient appearance of soluble protofibrils and ultimately to insoluble fibrils. The Abeta peptide is amphiphilic and accumulates preferentially at a hydrophilic/hydrophobic interface. Solid surfaces and air-water interfaces have been shown previously to promote Abeta aggregation, but detailed characterization of these aggregates has not been presented. In this study Abeta(1-40) introduced to aqueous buffer in a two-phase system with chloroform aggregated 1-2 orders of magnitude more rapidly than Abeta in the buffer alone. The interface-induced aggregates were released into the aqueous phase and persisted for 24-72 h before settling as a visible precipitate at the interface. Thioflavin T fluorescence and circular dichroism analyses confirmed that the Abeta aggregates had a beta-sheet secondary structure. However, these aggregates were far less stable than Abeta(1-40) protofibrils prepared in buffer alone and disaggregated completely within 3 min on dilution. Atomic force microscopy revealed that the aggregates consisted of small globules 4-5 nm in height and long flexible fibers composed of these globules aligned roughly along a longitudinal axis, a morphology distinct from that of Abeta protofibrils prepared in buffer alone. The relative instability of the fibers was supported by fiber interruptions apparently introduced by brief washing of the AFM grids. To our knowledge, unstable aggregates of Abeta with beta-sheet structure and fibrous morphology have not been reported previously. Our results provide the clearest evidence yet that the intrinsic beta-sheet structure of an in vitro Abeta aggregate depends on the aggregation conditions and is reflected in the stability of the aggregate and the morphology observed by atomic force microscopy. Resolution of these structural differences at the molecular level may provide important clues to the further understanding of amyloid formation in vivo.     

Does distance matter? Variations in alternative 3′ splicing regulation

Alternative splicing constitutes a major mechanism creating protein diversity in humans. This diversity can result from the alternative skipping of entire exons or by alternative selection of the 5′ or 3′ splice sites that define the exon boundaries. In this study, we analyze the sequence and evolutionary characteristics of alternative 3′ splice sites conserved between human and mouse genomes for distances ranging from 3 to 100 nucleotides. We show that alternative splicing events can be distinguished from constitutive splicing by a combination of properties which vary depending on the distance between the splice sites. Among the unique features of alternative 3′ splice sites, we observed an unexpectedly high occurrence of events in which a polypyrimidine tract was found to overlap the upstream splice site. By applying a machine-learning approach, we show that we can successfully discriminate true alternative 3′ splice sites from constitutive 3′ splice sites. Finally, we propose that the unique features of the intron flanking alternative splice sites are indicative of a regulatory mechanism that is involved in splice site selection. We postulate that the process of splice site selection is influenced by the distance between the competitive splice sites.     

Aggregate Clearance of α-Synuclein in Saccharomyces cerevisiae Depends More on Autophagosome and Vacuole Function Than on the Proteasome

Parkinson disease is the second most common neurodegenerative disease. The molecular hallmark is the accumulation of proteinaceous inclusions termed Lewy bodies containing misfolded and aggregated α-synuclein. The molecular mechanism of clearance of α-synuclein aggregates was addressed using the bakers' yeast Saccharomyces cerevisiae as the model. Overexpression of wild type α-synuclein or the genetic variant A53T integrated into one genomic locus resulted in a gene copy-dependent manner in cytoplasmic proteinaceous inclusions reminiscent of the pathogenesis of the disease. In contrast, overexpression of the genetic variant A30P resulted only in transient aggregation, whereas the designer mutant A30P/A36P/A76P neither caused aggregation nor impaired yeast growth. The α-synuclein accumulation can be cleared after promoter shut-off by a combination of autophagy and vacuolar protein degradation. Whereas the proteasomal inhibitor MG-132 did not significantly inhibit aggregate clearance, treatment with phenylmethylsulfonyl fluoride, an inhibitor of vacuolar proteases, resulted in significant reduction in clearance. Consistently, a cim3-1 yeast mutant restricted in the 19 S proteasome regulatory subunit was unaffected in clearance, whereas an Δatg1 yeast mutant deficient in autophagy showed a delayed aggregate clearance response. A cim3-1Δatg1 double mutant was still able to clear aggregates, suggesting additional cellular mechanisms for α-synuclein clearance. Our data provide insight into the mechanisms yeast cells use for clearing different species of α-synuclein and demonstrate a higher contribution of the autophagy/vacuole than the proteasome system. This contributes to the understanding of how cells can cope with toxic and/or aggregated proteins and may ultimately enable the development of novel strategies for therapeutic intervention.     

Growth‐driven displacement of protein aggregates along the cell length ensures partitioning to both daughter cells in Caulobacter crescentus

All living cells must cope with protein aggregation, which occurs as a result of experiencing stress. In previously studied bacteria, aggregated protein is collected at the cell poles and is retained throughout consecutive cell divisions only in old pole‐inheriting daughter cells, resulting in aggregation‐free progeny within a few generations. In this study, we describe the in vivo kinetics of aggregate formation and elimination following heat and antibiotic stress in the asymmetrically dividing bacterium Caulobacter crescentus. Unexpectedly, in this bacterium, protein aggregates form as multiple distributed foci located throughout the cell volume. Time‐lapse microscopy revealed that under moderate stress, the majority of these protein aggregates are short‐lived and rapidly dissolved by the major chaperone DnaK and the disaggregase ClpB. Severe stress or genetic perturbation of the protein quality control machinery induces the formation of long‐lived aggregates. Importantly, the majority of persistent aggregates neither collect at the cell poles nor are they partitioned to only one daughter cell type. Instead, we show that aggregates are distributed to both daughter cells in the same ratio at each division, which is driven by the continuous elongation of the growing mother cell. Therefore, our study has revealed a new pattern of protein aggregate inheritance in bacteria.     

An in silico model of the ubiquitin-proteasome system that incorporates normal homeostasis and age-related decline

Background  

The ubiquitin-proteasome system is responsible for homeostatic degradation of intact protein substrates as well as the elimination of damaged or misfolded proteins that might otherwise aggregate. During ageing there is a decline in proteasome activity and an increase in aggregated proteins. Many neurodegenerative diseases are characterised by the presence of distinctive ubiquitin-positive inclusion bodies in affected regions of the brain. These inclusions consist of insoluble, unfolded, ubiquitinated polypeptides that fail to be targeted and degraded by the proteasome. We are using a systems biology approach to try and determine the primary event in the decline in proteolytic capacity with age and whether there is in fact a vicious cycle of inhibition, with accumulating aggregates further inhibiting proteolysis, prompting accumulation of aggregates and so on. A stochastic model of the ubiquitin-proteasome system has been developed using the Systems Biology Mark-up Language (SBML). Simulations are carried out on the BASIS (Biology of Ageing e-Science Integration and Simulation) system and the model output is compared to experimental data wherein levels of ubiquitin and ubiquitinated substrates are monitored in cultured cells under various conditions. The model can be used to predict the effects of different experimental procedures such as inhibition of the proteasome or shutting down the enzyme cascade responsible for ubiquitin conjugation.     

A newly identified TSHβ splice variant is involved in the pathology of Hashimoto’s thyroiditis

Thyrotropin (TSH) is a protein that plays a key role in the control of thyroid function. TSH consists of a common α-subunit and a unique β-subunit; the latter is responsible for hormone specificity. A novel splice variant of human TSHβ was identified in 2009. To date, only the tissue distribution of the human TSHβ splice variant mRNA has been studied. Therefore, we aimed to characterize the protein translated from this splice variant. Salting-out, dialysis and concentration of serum proteins were followed by immunoprecipitation to identify the hTSHβ splice variant in serum. Stable CHO cell lines expressing the hTSHβ splice variant and V5-hTSHα were generated. After co-culture, co-immunoprecipitation was used to determine if the hTSHβ splice variant can dimerise with TSHα. We showed for the first time that the hTSHβ splice variant exists in human serum and dimerises with TSHα. To explore the relationship between the TSHβ splice variant and the pathogenesis of autoimmune thyroiditis, we assessed variations in the mRNA expression of the TSHβ splice variant in the peripheral blood leukocytes (PBLs) of Hashimoto’s thyroiditis (HT) patients using quantitative RT-PCR. We found that the mRNA expression levels of the TSHβ splice variant were higher in the PBLs of HT patients who were not undergoing prednisone therapy (n?=?10, P?<?0.0001) and in the PBLs of HT patients with a longer duration of illness (>18?months) who were undergoing prednisone therapy (n?=?5, P?=?0.023) than in those of the control group. This pattern was reversed in the PBLs of HT patients with a shorter duration of illness (<9?months) who were undergoing prednisone therapy (n?=?8, P?<?0.0001). Dexamethasone inhibition of the TSHβ splice variant mRNA expression occurred in a dose- and time-dependent manner. These results demonstrated that the TSHβ splice variant may participate in the pathogenesis of HT.     

To be or not to be toxic: aggregations in Huntington and Alzheimer disease

Insoluble aggregated proteins in Alzheimer disease and Huntington disease might not be pathogenic. Human studies have poor correlations between aggregates and clinical disease or pathology in these disorders, whereas mouse models have demonstrated that neuronal loss can occur in the absence of detectable aggregates. Furthermore, aggregates can exist in the absence of disease pathology in mice or symptoms in humans. Recent research suggests that soluble protein fragments, not insoluble aggregated proteins, are the toxic species in these disorders.     

Structure-Activity Analyses of β-Amyloid Peptides: Contributions of the β25–35 Region to Aggregation and Neurotoxicity

Abstract: The neurodegeneration of Alzheimer's disease has been theorized to be mediated, at least in part, by insoluble aggregates of β-amyloid protein that are widely distributed in the form of plaques throughout brain regions affected by the disease. Previous studies by our laboratory and others have demonstrated that the neurotoxicity of β-amyloid in vitro is dependent upon its spontaneous adoption of an aggregated structure. In this study, we report extensive structure-activity analyses of a series of peptides derived from both the proposed active fragment of β-amyloid, β25–35, and the full-length protein, β1–42. We examine the effects of amino acid residue deletions and substitutions on the ability of β-amyloid peptides to both form sedimentable aggregates and induce toxicity in cultured hippocampal neurons. We observe that significant levels of peptide aggregation are always associated with significant β-amyloid-induced neurotoxicity. Further, both N- and C-terminal regions of β25–35 appear to contribute to these processes. In particular, significant disruption of peptide aggregation and toxicity result from alterations in the β33–35 region. In β1–42 peptides, aggregation disruption is evidenced by changes in both electrophoresis profiles and fibril morphology visualized at the light and electron microscope levels. Using circular dichroism analysis in a subset of peptides, we observed classic features of β-sheet secondary structure in aggregating, toxic β-amyloid peptides but not in nonaggregating, nontoxic β-amyloid peptides. Together, these data further define the primary and secondary structures of β-amyloid that are involved in its in vitro assembly into neurotoxic peptide aggregates and may underlie both its pathological deposition and subsequent degenerative effects in Alzheimer's disease.